An Equal Opportunity Provider and Employer

VETERINARY SERVICES MEMORANDUM NO. 800.51 

TO: Veterinary Services Leadership Team 

 Directors, Center for Veterinary Biologics

 Biologics Licensees, Permittees, and Applicants

FROM: Jack A. Shere 

 Deputy Administrator 

SUBJECT: Additives in Administered Animal Biological Products 

I. PURPOSE 

This memorandum clarifies policies and procedures to comply with the requirements 

of title 9, Code of Federal Regulations (9 CFR), parts 103.2(b) and (c), 103.3(f) and 

(g), 112.2(a)(8), and 114.10. These regulations cover slaughter withholding periods 

for food animals when those animals have been administered biological products 

formulated with additives, and requirements for ingredients of animal origin. 

 

II.CANCELLATION 

 

This memorandum cancels Veterinary Services Memorandum No. 800.51, dated 

November 1, 2016. 

 

III. BACKGROUND 

A. Additives. Additives in veterinary biologicals may consist of adjuvants, 

carriers, inactivating agents, preservatives, or other ingredients added to 

cultures of microorganisms in the formulation of biological products used to 

treat animals. 

B. Adjuvants. Adjuvants are additives used to enhance the immunogenicity of 

the antigen. 

C. Authority and Consultation. 9 CFR 112.2(a)(8) prescribes a withholding 

period of not less than 21 days for food animals treated with veterinary 

biological products. APHIS may prescribe a longer withholding period on 

review of data regarding the use of additives in products used to treat 

animals. 

Animal and Plant

Health Inspection

Service

Veterinary Services

1400 Independence 

Ave, SW

Washington, DC

20250 for
VETERINARY SERVICES MEMORANDUM NO. 800.51 

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 An Equal Opportunity Provider and Employer

IV. SPECIFIC GUIDANCE REGARDING ADDITIVE SOURCES

A. Ingredients of Animal Origin 

1. Each lot of ingredient additive of animal origin used to prepare an 

administered biological product must be sterilized or tested as prescribed in 

9 CFR 113.53.

2. Ingredients of animal origin are sourced from the United States or 

countries considered free, low risk, or not affected with foreign animal 

diseases of concern and with negligible or controlled risk of bovine 

spongiform encephalopathy (BSE), according to APHIS’ Animal Disease 

Status designations, which may be viewed at:

https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-and-animal-productimport-information/animal-health-status-of-regions

The foreign animal diseases of concern to the Center for Veterinary 

Biologics (CVB) are: 

• Foot-and-mouth disease (FMD),

• African swine fever (ASF),

• Classical swine fever (CSF),

• Highly pathogenic avian influenza (HPAI), and 

• Newcastle disease (ND). 

No animal ingredients of avian origin may be sourced from a country if 

HPAI or ND is present in any region of the country. No ingredients of 

mammalian origin may be sourced from a country if FMD, ASF, or CSF 

is present in any region of the country. If an outbreak of any of these 

diseases occurs in countries providing ingredients of animal origin, 

consult the CVB for an assessment of risk. A risk assessment, including 

an analysis of the risk of contamination to licensed biologics, must be 

submitted to the CVB for review to justify using an ingredient of animal 

origin from a country that has a foreign animal disease of concern.

This guidance does not abrogate any other import requirements; all 

National Import and Export Services restrictions apply. 

3. Manufacturers must maintain a comprehensive list of all ingredients of 

animal origin used in production of biological products. This list should 

include the name of the material, the supplier, the country of origin, and 

the date of purchase of each lot. This list may be reviewed and 

certification of materials required at the time of inspection by the CVBInspection and Compliance (IC), or as requested by the CVB. 
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B. Ingredients Not of Animal Origin. Ingredients or other substances used in 

biological products not of animal origin must meet acceptable standards for 

purity and quality as specified in 9 CFR 113.50 and the filed Outline of 

Production. 

V. SPECIFIC GUIDANCE REGARDING PRESERVATIVES 

A. Antibiotics added to production media or at any subsequent stage of the 

manufacturing process are considered preservatives. The regulations at 9 CFR 

114.10 specify the maximum permissible amounts and combinations of 

antibiotics authorized for use as preservatives in veterinary biologics. The 

maximum allowable concentrations of antibiotics listed are expected to inhibit 

growth of common susceptible bacterial contaminants, and a definite genetic 

determinant for resistance would be necessary for survival at these 

concentrations. Conventionally, amphotericin B has been dissolved in a small 

volume of dimethyl sulfoxide (DMSO). Amphotericin B dissolved in DMSO 

is permitted as stated in the Amphotericin B general requirements in 9 CFR 

113.10.

B. Antibiotics used in virus seed stock purification are not restricted as to amount

if carryover into the final product is controlled and specified in the Outline of 

Production. The CVB considers carryover to be controlled if the antibiotics 

used in seed stock purification are added to the seed stock culture in 

concentrations no greater than generally accepted inhibitory levels for 

susceptible contaminants and/or the resultant culture media is decanted or 

filtered. 

 

C. A request to use an antibiotic or antibiotic combination not specified in 9 CFR 

114.10(b) or (c) as a preservative may be considered under certain conditions. 

A request for use should include the reason why the antibiotic or antibiotic 

combination is needed, a risk assessment, the expected levels of the antibiotic 

in the final product, and data indicating the residual antibiotic level in tissues 

of animals to which the product is administered. 

VI. SPECIFIC GUIDANCE REGARDING SLAUGHTER WITHDRAWAL 

PERIODS/ ADJUVANT APPROVALS FOR INJECTABLE PRODUCTS 

A. Describe the Adjuvant. All product licensing packages should include a 

detailed description of the formulation of any adjuvants included in the 

product. This information should be provided early in the licensing process. In 

general, all product license applications should contain the following 

information: 

1. Generic name of adjuvant (and trade name, if applicable). 
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 An Equal Opportunity Provider and Employer

2. Chemical composition of adjuvant (list all ingredients and proportions). 

3. Amount of completed/total adjuvant per dose of product and dose volume 

of product. Include the percentage of adjuvant per dose. 

4. Animal species in which product is to be used. 

5. Route of administration. 

6. Information regarding source, grade, and quality of each ingredient. 

7. Any tests performed on ingredient lots and the finished adjuvant prior to 

use. 

8. Proposed slaughter withdrawal period and supporting data. 

9. Other products for which the adjuvant has been approved, if applicable. It 

is understood that in some cases, adjuvants are purchased from another 

firm; and, therefore, some of the data listed may be proprietary 

information of the supplier and may not be available to the recipient. In 

this case, it is acceptable for the recipient to request that the supplier 

submit the necessary information to the CVB in a manner that protects 

confidential business information. 

B. CVB Review of Adjuvant Description. The adjuvant description and 

composition will be compared to historic data on file at the CVB. Comparison 

of the adjuvant description and composition with historic data may indicate 

that these data, in addition to satisfactory field safety trial results, are adequate 

to approve the use of the adjuvant in the new product. 

C. Additional data may be required for previously-approved adjuvants used in 

new ways. To approve new adjuvants or previously-approved adjuvants used 

at increased levels, administered via different routes of administration, or 

proposed for shorter withdrawal periods, results of an injection site study in 

the host animal may be requested. The following guidelines should be 

considered when conducting an injection site study: 

1. Submit a protocol for comment. The protocol should include key dates to 

allow observation by CVB personnel. 

2. At least 10 animals of the minimum age of the target species should be 

included for adjuvants used in food-producing species other than fish and 

poultry. 
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a. The proposed product should be administered as per the proposed label 

directions, and the withdrawal period should be evaluated after the last 

dose is administered. 

b. Typically, the new adjuvant should be compared to the productmatched placebo by injecting the new product on one side of the 

animal, and the placebo at the same site on the other side of the 

animal. For each animal, assign the products randomly to the two

sides. Comparing more than two products within an animal often calls 

for a more complex randomization plan that should be discussed with 

the CVB. Exceptions to using product-matched placebos may be 

considered. This issue will be addressed in the review of the protocol 

by the CVB. 

c. The injection sites should be examined grossly by a veterinarian or 

veterinary pathologist blinded to the study, and results of the gross 

pathologic examination should be included in the report. The 

veterinarian/pathologist should also collect appropriate tissues for 

histopathology, including the injection sites (adjuvant and placebo). 

Samples for histopathology should be collected at the time of the 

proposed slaughter withdrawal period (i.e., if the proposed slaughter 

withdrawal period is 21 days, samples should be collected 21 days 

after injection). Photographs of any gross lesions at the injection site 

should be included in the final report. 

d. Tissue samples should be examined histologically by a board certified 

pathologist who has no knowledge regarding the products used in the 

study; photomicrographs of histologic changes present at the injection 

site should be included in the final report. The histologic evaluation is 

conducted to evaluate and ensure that the local inflammatory response 

is consistent with resolution of the expected physiologic/immunologic 

response to foreign material in the respective tissue. For example, 

observations of ongoing inflammation that are inconsistent with 

resolution may require additional evaluation and/or longer withdrawal 

periods. 

e. Collecting biopsies after local anesthesia, or using alternate methods to 

salvage study animals, may be acceptable. 

3. For poultry, use at least 10 birds of minimum age of the target species. 

The new adjuvant should be compared to a product-matched placebo by 

injecting the new product on one side of the animal, and the productmatched placebo at the same site on the other side of the animal. Injecting 

the same bird with the placebo and the proposed product is not required if 

impractical or if two doses would cause deleterious effects not related to 
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 An Equal Opportunity Provider and Employer

adjuvant safety. (For example, if day-old chicks are inoculated, it would 

be acceptable to include a group of birds inoculated with product and a 

group of birds inoculated with matched placebo.) As with injection site 

studies for other species of animals, samples should be collected and 

evaluated as per sections VI.C.2.b and VI.C.2.c of this document. 

4. At least 20 fish of minimum age/size of the species in which the product is 

to be used should be included for injection site studies in aquatic species 

meant for human consumption. The study may be done using the same 

dose as per label recommendations, or at double the dose recommended on 

the label. 

a. Many adjuvants cause some degree of tissue adhesion and 

pigmentation in the abdominal cavity of fish when administered 

intraperitoneally. The Speilberg Scoring System, based on the size and 

density of the adhesion, should be used to analyze data from aquatic 

species. The study will be evaluated for degree of tissue pigmentation 

and adhesion in the abdominal cavity, as well as residual vaccine or 

vaccine components in the abdominal cavity.

b. Residual vaccine or vaccine components present at slaughter in edible 

portions of fish are not acceptable. 

c. Injection sites should be examined grossly by a veterinarian, veterinary 

pathologist, or fish health specialist blinded to the study, and results of 

the gross pathologic examination should be included in the report. 

Samples for histopathology should also be collected and evaluated, as 

described in section VI.C.2.C of this document. 

5. For animals not meant for human consumption (e.g., dogs, cats), the 

results of an acceptable field safety study are adequate to demonstrate 

safety of the adjuvant. 

6. Summarize results from infection site studies with descriptive statistics. 

Avoid statistical inference that may not be supported by the study size or 

design.

7. Data generated from field safety or efficacy studies might support 

slaughter withdrawal periods of adjuvanted products when an injection 

site study has been accepted using a different antigen. Data will be 

considered on a case-by-case basis. 

8. A single injection site study may be used to support the slaughter 

withdrawal period of not only a specific adjuvant, but also other adjuvants 
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 An Equal Opportunity Provider and Employer

formulated with lesser amounts of the same components, when used in the 

same species of animal and administered by the same route of inoculation. 

9. Injection site study results such as the following would be considered 

unacceptable: 

a. Significant gross lesions (e.g., purulent or caseous material) present at 

the proposed slaughter withdrawal time. 

b. Indications of active inflammation at the time of slaughter. 

c. Lesions observed histologically that are inconsistent with gross lesions 

observed. 

D. Additional information that may be included as supporting data in an injection 

site study report: 

1. Summary of other studies (e.g., peer-reviewed publications, relevant 

internal reports) that contain pathological assessments of the experimental 

product when administered to the target species. 

2. Data showing the time required for the outward resolution of any injection 

site reactions (obtained from the injection site study report, field safety 

report, and/or efficacy study report of the product to be licensed). 

3. Results of safety studies in laboratory animals. 

E. Additional considerations for novel adjuvants or other additives. For new 

additives, including unique adjuvants that have not been included in 

previously approved products, the following information may be required: 

1. Toxicological profile, which should include: 

a. Any information relative to the listing of the additives on lists of 

approved additives (e.g., Generally Regarded as Safe (GRAS), 

Drinking Water Standards). Provide a copy of the Material Safety Data 

Sheet (MSDS) or reference the MSDS number for each additive, if 

available. 

b. The results of toxicological studies to determine the local and/or 

systemic effects of the additive on laboratory animals, if available. 

c. Summary of any oral/acute testing of the additive in target and nontarget species. 
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 An Equal Opportunity Provider and Employer

d. Summary of any information available regarding the metabolism of the 

additive. 

e. Any information available regarding the carcinogenicity of the additive. 

f. Any known reactivity of each additive. 

g. Pharmacological activity of each additive. 

2. Human exposure profile, which should include: 

a. Estimate of the total volume/mass of the additive that will be 

administered to the target animal under the proposed instructions for 

use. 

b. Estimate of the human consumption/exposure to each additive. 

c. Levels of residue in tissue at proposed withdrawal period. The levels 

should not exceed the Food and Drug Administration (FDA) tolerances 

for food, if available. 

d. FDA tolerance level established for each additive (cite source). 

VII. EXEMPTIONS TO SLAUGHTER WITHDRAWAL PERIOD 

REQUIREMENTS 

A. No slaughter withdrawal statement is needed for a non-parenteral (e.g., oral, 

intranasal, immersion) product used solely for neonatal animals that do not 

enter the food chain. 

B. Non-parenteral biologics used for food-producing animals entering the food

chain typically are assigned a slaughter withdrawal period of 21 days. 

VII. IMPLEMENTATION

APHIS expects to implement this guidance within 2 years from the date of this 

memorandum.